The production of large quantities of relatively pure polypeptides and proteins is important for the manufacture of many pharmaceutical formulations. For production of many proteins, recombinant DNA techniques have been employed in part because large quantities of exogenous proteins can be expressed in host cells.
Plasmin, the principle fibrinolytic enzyme in mammals, is a serine protease with trypsin-like specificity that is derived from the inactive zymogen precursor plasminogen circulating in plasma. Plasminogen itself is a 790 amino acid polypeptide having an N-terminus glutamate residue. Plasminogen activators such as streptokinase, tissue plasminogen activator (tPA) or urokinase will cleave the single-chain plasminogen molecule to produce active plasmin at the Arg560-Val561 peptide bond. The resulting two polypeptide chains of plasmin are held together by two interchain disulfide bridges. The light chain of 25 kDa carries the catalytic center and is homologous to trypsin and other serine proteases. The heavy chain (60 kDa) consists of five triple-loop kringle structures with highly similar amino acid sequences. Some of these kringles contain so-called lysine-binding sites that are responsible for plasminogen and plasmin interaction with fibrin, α2-antiplasmin, or other proteins.
Plasmin as a potential thrombolytic agent has numerous technical difficulties. These difficulties include the challenge of preparing pure plasmin that is relatively free of functional traces of the plasminogen activator used to generate plasmin from its inactive precursor, plasminogen. Preparations of plasmin are typically extensively contaminated by plasminogen activator, streptokinase or urokinase, and the thrombolytic activity has been, therefore, attributed to the contaminating plasminogen activators rather than to plasmin itself. The contaminating plasminogen activators could also trigger systemic bleeding other than at the targeted site of thrombosis. Another important technical factor limiting clinical use of plasmin is that plasmin, as a serine protease with broad specificity, is highly prone to autodegradation and loss of activity. This circumstance provides severe challenges to the production of high-quality plasmin, to the stable formulation of this active protease for prolonged periods of storage prior to use, and to safe and effective administration of plasmin to human patients suffering from occlusive thrombi.
Preparative isolation of recombinant plasminogen or plasmin prepared from recombinant plasminogen resulting in pharmaceutical purity and sufficient yield has eluded the art. Thus, there is need for compositions and methods for preparing a recombinant plasminogen and plasmin prepared from recombinant plasminogen activated by a plasminogen activator.